a sex-linked gene controlling the onset of … · a sex-linked gene controlling the onset of sexual...

A SEX-LINKED GENE CONTROLLING THE ONSET OF SEXUAL MATURITY IN FEMALE AND MALE PLATYFISH

(XZPHOPHORUS MACULATUS) , FECUNDITY IN FEMALES AND ADULT SIZE IN MALES

KLAUS D. KALLMAN AND VALERIE BORKOSKI

Osborn Laboratories of Marine Sciences New York Aquarium, New York Zoological Society, Brooklyn, New York 11224

Manuscript received August 24, 1977 Revised copy received December 22, 1977

ABSTRACT

A sex-linked gene, P, controls the onset of sexual maturity in the platyfish, Xiphophorus maculatus. The activity of this gene is correlated with the age and size a t which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active. Immature fish of all genotypes grow at the same rate; however, as adults, males with “early” genotypes are significantly smaller than males of “late” genotypes, since growth rate declines strongly under the influence of androgenic hormone. Five alleles, PI . . . P5, have been identified from natural populations that under controlled conditions cause gonad maturation between eight and 73 weeks. PlPl males become mature at eight weeks and 21 mm, P2Pa and P V J males between eleven and 13.5 weeks and 25 to 29 mm, and P4P4 males at 25 weeks and 37 mm. Since P5 is X-linked, no males homozygous for P5 could be produced. The difference between Pa and P3 is largely based upon their interaction with P5. PJP5 males mature at 17.5 weeks and 33.5 mm and P2P5 males at 28 weeks and 38 mm. The rate of transformation of the unmodified anal fin into a gonopodium, which is under androgenic control, is directly related to the age at initiation of sexual maturity, ranging from 3.2 weeks in PIPI males to seven weeks in Pap5 males. These differences may reflect different levels of circulating gonadotropic and androgenic hormones.-In two genotypes of females, initiation of vitellogenesis was closely correlated with size and this critical size was inde- pendent of age (e.g., 21 mm for PIP’). In a third genotype (PIP) the minimum size for vitellogenesis decreased with increasing age, so that females would mature as early as eleven weeks, provided they had attained at least 29 mm, but at 25 weeks even females as small as 23 mm possessed ripe gonads. For P5P5 females, which become mature between 34 and 73 weeks of age, there is no correlation between size and initiation of vitellogenesis. In all four genotypes of females examined, egg number is strongly correlated with size, but the regression of egg number on standard length is distinct for each genotype. Late maturation of P5P5 females is not offset by an increased number of eggs; for this genotype there is a strong negative correlation between age and number of eggs. Heterozygous fish always mature later than those homozygous for the “earlier” allele. The site of action of the P locus could be the pituitary gland, the hypothalamus or higher centers of the brain where peripheral information is transduced into an appropriate signal required for the activa-

Genetics 89: 79-119 May, 1978.

80 K. D. KALLMAN AND V. BORKOSKI

tion of the hypothalamus-pituitary-gonadal axis. The P gene could also control the peripheral information. The platyfish may be a useful model to test theories concerning the evolution of life history strategies.

HE onset of gonad maturation in the platyfish. Xiphophorus macutatus, is Tcontrolled by a sex-linked gene, P ( KALLMAN, SCHREIBMAN and BORKOSKI 1973). In this paper we present evidence for the existence of at least five alleles at this locus. Depending upon genotype, male and female platyfish initiate maturity between five and 73 weeks of age. A certain number of fish of one genotype apparently never became sexually mature. The onset of vitellogenesis is closely correlated with size in some genotypes, with age being relatively unim- portant; in other genotypes either age alone or both age and size are important factors. The number of ripe ova for a given size is also dependent upon the P genotype. Because of allelic interactions, the phenotypes of heterozygotes cannot always be predicted from that of the homozygous parental genotypes.

This genetic polymorphism has other far-reaching effects. The growth rate of females and immature males, regardless of genotype, is the same. But since the growth rate of male platyfish declines sharply under the influence of androgenic hormones, males with the early genotypes remain relatively small, while those with late genotypes grow significantly larger.

All of the parameters influenced by the P locus are key traits for life-history tactics (STEARNS 1976). Based upon cross-species comparisons, it seems that in expanding populations or in those in a fluctuating environment, early maturity, large number of progeny and increased reproductive effort is favored, while in stable o r declining populations the opposite traits are present. However, direct experimental evidence concerning whether a certain set of traits is selected for under particular circumstances is not available. The platyfish now affords a simple genetic system to test some of the theories concerning the evolution of life-history strategies.

Moreover, by providing the experimenter with fish of early- and late-maturing genotypes that are identical in all other respxts, the platyfish represent a model system that allows us to study separately the effects of age and size on the onset of sexual maturity.

The activity of the P gene is correlated with the age and size at which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active (KALLMAN and SCHREIBMAN 1973). The initiation of gonadotrop function coincides with the massive growth of the gonads in both sexes, vitellogenesis in females and spermatogenesis and development of secondary sex char- acters in males. Platyfish are born with a pituitary gland in which all the cell types present in mature individuals can already be identified, with the exception of the gonadotrops (cyanophils) in the peripheral meso-adenohypophysis (SCHREIBMAN 1964). In the absence of functional gonadotrops. oocytes develop as far as the oil-droplet stage and male germ cells as far as spermatogonial stage I1 ( SCHREIBMAN and KALLMAN 1977).

A similar genetic polymorphism has been analyzed in two populations of

GENETIC CONTROL OF SEXUAL MATURITY 81

Xiphophorus pygmaeus. It differs from that of X . maculatus in being restricted to males, and differences in size at initiation of maturity are accompanied by differences in growth rate (KALLMAN, unpublished). Males of several other species of poeciliid fishes exhibit intraspecific differences in adult size of comparable order of magnitude as those of X . maculatus and X . pygmaeus, raising the possibility that the P locus may be widespread in this family (SCHREIBMAN and KALL- MAN 1977).

MATERIALS A N D METHODS

X . macuhtus occurs in the lowlands of southern Mexico, from the Rio Jamapa, Veracruz, across northern Guatemala (Peten district, Aha Verapaz) into Belize. All crosses were performed with known descendants of fish collected by Genetics Laboratory personnel during the last 35 years from natural populations of X . maculntus in Mexico (Rio Jamapa, Rio Papaloapan, Rio Coatzacoalcos) and in Belize (Belize River). The genetic analysis was facilitated by the presence of several sex-linked, codominant pigment factors that were closely linked to the pituitary locus, P, and that served as genetic markers (KALLMAN 1973). Because of the existence of three sex chromosomes, W , X and Y , in most natural populations of X . maculatus, the sex genotype of females may be either W Y , W X or X X and that of males XY, or YY (KALLMAN 1973). Since the beginning of gonad maturation is easily influenced by many variables, every effort was made to standardize conditions under which the experimental fish were raised, SO that differences or similarities in age of gonad maturation between genotypes could be attrib- uted to sex-linked P factors and not to genetic background or rearing conditions. For this reason, we have compared mainly experimental genotypes that were of the same sex, that were members of the same brood and cross, and that were raised under identical conditions. Experimental groups of males consisted either of two or four genotypes. In the first case (e.g., from X X 0 0 X X y 8 8 ), the two genotypic classes under consideration always shared one sex chromosome; either both classes of males were heterozygous (e.g., X - A Y-B versus X-C Y-B, where A, B and C stand for different pigment and P factors) or one of the classes was homozygous ( X - A Y-B versus X-B Y-B). In the second case, the four genotypic classes of males were derived from crosses of the type X X 0 0 x Y Y 8 8 in which all four sex chromosomes were marked by different pigment factors. Note that the four classes can be arranged in a series in which only one sex chromosome has been changed at one time. If these sex chromosomes also carried different P factors, the four pigment classes might also differ with respect to age and size a t maturity. A significant difference ( P < 0.05) in age of sexual maturity between genotypes within an experimental group was taken as evidence that the P factors on the sex chromosomes not shared by the two, or four, classes of males were not identical.

As a result of sex-linkage and the low frequencies of crossing over between the sex-differential segment, the pigment loci and the pituitary gene, P, a number of genotypes could only be produced either in males o r females. The existence of two sex chromosomes, W and X , determining femaleness permitted the synthesis of females heterozygous for Y-linked P factors, but such females could not be made homozygous for them. Conversely, males might be homozygous for Y-linked P factors ( Y Y ) , but for factors on the X chromosomes they could only be heterozygous. This does not preclude the possibility that certain P factors may be present on both X and Y chromosomes.

The origins of the sex chromosomes used in the current study and their marker (pigment) genes have been listed in Table 1. X-Spl, X-Dr, and Y-Sr are the sex chromosomes of the Jamapa stocks (Jp) derived from fish collected in 1939. Y-Br, Y-Zr and W-+ came from the Belize population (Bp) collected in 1966 (KALLMAN 1970a), and X-NI originated from a fish taken in 1969 at a site a few kilometers from the 1966 location. Y-N*, Y-SpO and Y-Ar can be traced to fish collected from a location near the mouth of the Rio Coatzacoalcos (Cp-2840) in


TABLE 1

Sex chromosomes of fish used in the current study

Stock Sex chromosomes* Piement Dattem P factor

Jamapa Jp 163 B Jamapa Jp 163 A Jamapa Jp 163 A,B Belize BP

Belize Belize Belize Coatzacoalcos Coatzacoalcos Coatzacoalcos Coatzacoalcos Papaloapan

BP BP BP c p 2840 Cp 2840 Cp 2840 Cp 2840 Pp 2842

x-Sp’ X-Dr(Sd) Y-Sr X-(CPo) NI

w-+ Y-Ir Y-Br Y-spg Y-N’ Y-Ar w-+ x-Iy

spot-sidedb red dorsal fin, (spotted-dorsal) stripe-sided (caudal peduncle orange),

wild type red iris red body spot-sided black-banded red anal fin wild type yellow iris

black-bandedc

P’ PI PZ

P5

not determined PJ P4 P’ P’

not determined not determined not determined

a The patterns of the genes in ( ) have been used as markers only in one pedigree. b The spot-sided patterns of the different populations are caused by different allelic S p factors.

Some populations contain two o r more Sp factors. They have been arbitrarily numbered (KALLMAN 1975).

c The black-banded Datterns of the different Douulations are caused by different N factors. They have been arbitrarily numbered (KALLMAN 19?5j.

1971. X-Iy was taken from the Papaloapan stock (Pp-2842), which was derived from fish collected 12 km west of Ciudad Aleman (Rio Papaloapan drainage) in 1971.

Linked to Dr is Sd, but since Sd exhibited low penetrance in hybrids between different platyfish populations (KALLMAN 1970b), it was used only once as a genetic marker (see pedigree 3153 below). In this pedigree, X-Dr X-NI and X-NI X-NI females were compared with each other, but since a second pigment factor, CPo (caudal peduncle orange), linked to NI, extended its phenotypic effect into the dorsal fin when introduced into a gene pool largely derived from Jamapa, Dr could not be used as a marker.

The sex chromosomes listed in Table 1 were selected from the many sex chromosomes present in the platyfish stocks of the Genetics Laboratory. The Jamapa strains had been involved in a large number of different experiments since 1939. Any additional genetic information uncovered, especially one as important as the onset of gonadotrop activity, would be highly relevant for many different biological inquiries. The Cp and Bp stocks were used because preliminary observations suggested that some of their sex chromosomes might be carrying the “earliest” and ‘‘latest’’ P factors known. X - I y of Pp was chosen because it provided the only suitable X-linked marker gene to distinguish between homozygous and heterozygous N’ females.

Since the onset of gonadotrop differentiation and function can be ascertained only after a considerable amount of histological work, we have relied instead upon the metamorphosis of the anal fin into a gonopodium, an event that can be detected in the living fish and is known to be closely correlated with testis and gonadotrop development (SCHREIBMAN and KALLMAN 1977). This fin transformation proceeds through six clearly defined stages (KALLMAN and SCHREIBMAN 1973). The anal fin in stage one is fan-shaped, and this condition is typical for males with an undeveloped gonadotropic zone. Coincident with the initiation of gonadotrop differentiation and function, the testis begins to secrete androgens that in turn cause the elongation of the third, fourth and fifth anal fin rays (GROBSTEIN 1948). This is gonopodial (anal fin) stage two and represents the first external manifestation that a male has entered the process of gonad maturation. Gonopodia that have reached stage six are fully differentiated, and males with such

GENETIC CONTROL O F SEXUAL MATURITY 83

gonopodia are considered sexually mature. It should be noted, however, that spermatozoa and a few spermatozeugmata are already present in the developing testes of males in gonopodial stages two and three, respectively (KALLMAN and SCHFLEIBMAN 1973). Since the time required for complete anal fin metamorphosis was also controlled by the P genotype, we have listed for each genotype the mean age at both stage two and stage six. Depending upon genotype, the gonopodial stage was examined under lox magnification every other day M once a week.

Sexual maturity in females can be ascertained only by autopsy, since no secondary sex char- acters are known. Females were considered sexually mature when their ovaries contained at least one large yolky egg. The number of such ripe eggs was determined and recorded.

Fish were raised either in isolation (treatment i) by placing them at the age of ten days into 6.5- or 17-liter aquaria (except ped. 3131, see below) or they were reared under mass culture conditions (treatment m) that were highly variable and range from two fish in a 17-liter tank to more than a dozen fish in 33 liters. Nevertheless, all the fish of a given experi- ment group were always exposed to exactly the same treatment. Different broods of the same cross were often treated differently. Because of the large number of tanks required, relatively few experimental groups could be raised in isolation. Large broods were divided when ten days old into equal lots and distributed into two or three aquaria. Results obtained from such divided broods did not differ from each other; therefore, these data have always been combined. Observa- tions made on different broods of the same cross have been listed separately even when the fish were exposed to the same treatment, except in ped. 3538 for which the data from several small broods were combined.

One factor over which we had no control concerned the number of young per brood and their developmental stage at birth. The fish of a few broods were born with a prominent yolk sac; in others this had already been resorbed at birth. Members of large broods often seemed smaller in size and weaker at birth than fry of small broods. The fish of the second brood of ped. 3131 were a case in point. This brood of 55 young (23 9 9 ,32 8 8 ) was born in a 17-liter aquarium and maintained there for ten days. This represented extremely crowded conditions, which may have affected growth and eventually the onset of sexual maturity.

All fish were reared and maintained in aged aquarium water that has been kept in con- tinuous use since 1939; water lost through evaporation was made up biweekly by the addition of a small amount of New York City tap water. The tanks contained plants and gravel, but no aeration or filtration was used. Approximately every 15 months, the organic debris that accumu- lated at the bottom of the aquarium was siphoned off. Natural light entered the laboratory through a skylight equipped with baffles and adjustable louvers to block out the direct rays of the sun. Between September and March, incandescent lamps provided additional light until 9:OO p.m. The temperature of the aquarium water averaged 23". Platyfish (strain Jp 163A) also become mature in less than 14 weeks in an environmental chamber at 23" on an 8/16 light-dark cycle (KALLMAN, unpublished). Fish were fed three times daily ad libitum. Alter- nately, they were given dried shredded shrimp or liver paste (GORDON 1950) in the morning or afternoon and live brine-shrimp nauplii at noon. Fish were measured along the flank from snout to bifurcation of the caudal blood vessel (standard length) with calipers to the nearest half millimeter. All measurements were performed with fish anesthetized with MS 222 before being measured.

Sex and the color pattern of the offspring could not be recognized until several weeks after birth, and the decision as to the kind of treatment for a given brood was made before any young were born. All fish of this study were eventually preserved in 10% formalin and deposited in the morgue of the Genetics Laboratory for future reference.

RESULTS

I. Males Belize stock: The discovery that both the onset of sexual maturity and, indi-

rectly, the adult size of males were determined by a sex-linked gene, was made

84 K. D. KALLMAN A N D V. BORKOSKI

in the Belize stock (KALLMAN, SCHREIBMAN and BORKOSKI 1973). Males of this strain were homogametic, YY, the Y chromosomes being marked by either Zr (red iris) or Br (red body), and females were heterozygous, W-4- Y-Zr or W-4- Y-Br. Under mass culture conditions, males homozygous for Br took twice as long to reach sexual maturity (average 26.5 weeks) and were significantly larger than males homozygous for Zr (Table 2). Heterozygotes were intermediate for both age of sexual maturity and adult size. The difference between the three genotypes was present regardless of rearing conditions. Only five males of the Belize stock have been raised in isolation (ped. 3373). Under these conditions, limited as these data are, the males reached maturity slightly earlier and at a somewhat larger size (Table 2).

Jamapa stocks: The two Jamapa stocks, Jp 163 A and 163 B, are homogametic in females (X-Dr X-Dr and X-Sp' X-Sp') and heterogametic in males (X-Dr Y-Sr and X-Sp' Y-Sr) . The following experiments were performed to determine whether the P factors on the X chromosome marked by Sp' and on the Y chromosome marked by Sr were identical. For this test we have produced X-Sp' Y-Sr and Y-Sr Y-Sr males within the same pedigree by taking advantage of a rare X-Sp' Y-Sr female that arose spontaneously in a previous pedigree (ped. 3080, see below). Exceptional X Y females, which are occasionally found in the Jamapa stocks and in fish derived from them (ANDERS and ANDERS 1963; KALLMAN 1965; MACINTYRE 1961), are fully functional. From a cross of a X-Sp' Y-Sr 0 (ped. 3080) x X-Sp' Y-Sr 8 , 31 Sp' females, 4.0 Sp' Sr males and 20 Sr males were obtained (ped. 3131). Sp' Sr males attained stage six two weeks earlier than their Sr Sr sibs, and this difference was present regardless of rearing conditions (Table 3). Sp' Sr males also entered stage two about ten days earlier than Sr males. The differences in age of sexual maturity were reflected in adult size. Males homozygous for Sr were significantly larger than Sp' Sr males.

Additional evidence that the P factors linked to Sp' and Sr cannot be identical was provided by pedigree 3134 in which X-Sp' Y-Zr males were compared with Y-Sr Y-Zr males. The Sp Zr class matured earlier (78 days) and were smaller (21 mm) than the Zr Sr class (86.3 days, 22.4 mm) .

Diflerence between X-Spl of J p and Y-Ir of Bp: The results of pedigrees 3229 and 3200 in turn demonstrated that the P factors linked to X-Sp' and Y-Zr cannot be identical. Sp' Zr males matured significantly earlier and were smaller than homozygous Zr males (Table 3).

Comparison of Y-Sr of Jp with Y-Ir of Bp: The P factors linked to Y-Sr and Y-Zr had similar effects on gonad maturation (peds. 3131, 3134, 3229, 3200). Sp' Sr and Sp' Zr males matured nearly at the same age. Moreover, the differences between Sp' Zr and Sr Zr males were quite similar to those between Sp' Sr and Sr Sr males or between Spl Zr and Zr Zr males (Table 3). The possibility that the P factors on Y-Sr and Y-Zr might be identical has been examined in peds. 3346,3230 and 3276 (Table 3) .

A test of Sp' Zr against Sp' Sr males within pedigree 3346 was inconclusive. No significant difference was detected between the two classes in the first brood.


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86 K . D. K A L L M A N A N D V. BORKOSKI

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In the second brood, Sp' Sr males matured significantly earlier than their Sp' Zr sibs. This difference was apparent as. early as seven weeks after birth when all but two Sp' Sr males had entered stage two, whereas only two Zr males had reached this stage. The two kinds of males did not differ in size. Among the Zr males, however, there was one "runt" that measured only 19 mm at 14 weeks; the next smallest Zr male was 25 mm. When this runt was eliminated from the size calculations, the difference between the two genotypes became significant (Sp' Zr 8 8 , 26.4 +. 0.5 mm, p = 0.01). When the data of the two broods were combined, the two classes of males did not differ significantly in the age at maturity ( p = 0.1).

The same problem was investigated in pedigrees 3204 and 3230 in which homozygous Sr Sr males were compared with Sr Zr males. Only a small number of progeny was obtained from each cross. The difference between the two genotypes was not significant in either case. Nevertheless, as in pedigree 3346, Zr males were again somewhat larger and matured later than their non-Zr sibs, regardless of treatment. In a fourth pedigree, 3276, Y-Br Y-Sr males were com-

TABLE 4

Genetic history and parental genotypes of all pedigrees with NI

P, females P, males* Pedigree Strain Strain X Y

of or or or progeny pedigree X X pedigree Y Y*

2460 Jp 163 A Dr Sd Dr Sd BP NlCPo CPo 2744 2460 N' Dr Jp 163 A Dr Sr 3060 2744 N' Dr Bp 2918* Ir Br 3065 2744 N' Dr Bp 2918* Ir Br 3073 2744 N' Dr Jp 163 A Dr Sr 3080 Jp 163 B Sp' Sp' 2744 N' Sr 3145 3080 N' SP' Jp 163 A Dr Sr 3149 3080 N' SP' Jp 163 A Dr Sr 3153 3073 N'CPo DrSdb 3065 N'CPo Ir 3209 3080 N' SP' Bp 31(E1* Ir Br 321 1 3080 N' SP' Bp 3030* Ir Br 3295 Pp 3143 IY IY 3145 N' Sr 3435 3295 N' IY 3145 N' Sr 3444 3295 N' IY 3145 N' Sr 3445 3295 N' IY 3145 N' Sr 3446 3295 N' IY 3146 N' Sr 3447 3295 N' IY 3146 N' Sr 3471 Jp 163 B Sp' Sp' 3145 N' Sr 3537 3471 N' SP' Jp 163 B Sp' Sr 3538 3471 N' SP' Jp 163 B Sp' Sr 3634 3538 N' SP' Jp 163 A Dr Sr 3635 3538 N' SPl Jp 163 A Dr Sr 3636 3538 N' SP' Jp 163 A Dr Sr

a YY males are identified by an *. b Sd is linked to Dr and CPo is linked to NI. Sd and CPo were important only as genetic markers

in this pedigree, see text; Sd and CPo were therefore omitted from all other pedigrees.


pared with Y-Br Y-Zr males. The males with Sr reached stage 6 two weeks earlier than their sibs with Zr ( p < 0.01) and averaged 2 mm smaller (p = 0.01). The males of ped. 3276 had been distributed over four aquaria (see footnote n, Table 3). We have performed an ANOVA to determine whether the males of each genotype behaved similarly in each tank. For Br Sr males, this gave an F ratio of 1.306 (treatment df 3, MS 2.444, error df 12, MS 1.866) and for Br Zr males an F ratio of 0.603 (treatment df 3, MS 4.190). Neither F ratio was significant.

Summarizing these observations, we find that in four pedigrees comprising five broods, two kinds of treatment and a comparison of three sets of genotypes, Zr males always reached stage two later than Sr males, but that this difference was not always statistically significant. These observations suggest that the P factors linked to Zr and Sr are not identical, but are similar in their effects. TWO allelic combinations will be discussed below that clearly show these two P factors to be different.

The lute P factor on X-N1 of Bp: This chromosome can be traced to Bp male 3294-11, X-N'CPo Y-CPo, collected in Belize in 1969. Since that time this chromosome has been introduced through a series of backcrosses into the Jamapa stocks. The genetic history of all experimental N' males and females (see below) has been listed in Table 4 to document the fact that sexual maturity of N' fish and their non-N1 siblings has been studied on a variety of genetic backgrounds. Ped. 2744, in which the late maturation of N' Sr males was f i s t noted (KALL- MAN and SCHREIBMAN 1973), represented the first backcross generation. In this study (Table 5) we compared N 1 Sr males with their Dr Sr sibs in ped. 3073 (2nd Bc generation and with Sp' Sr males in peds. 3537 and 3538 (5th Bc generation).

Sr males without N' matured between 12 and 15 weeks of age, whereas N' Sr males did not attain stage six until 27 to 32 weeks (Table 5 ) . This difference of 14 to 18 weeks, which is the largest discovered to date between two genotypes of males within a single pedigree, occurred in all three pedigrees and experimental groups regardless of whether the fish were raised in isolation or in mass cultures. The P genotype also affected the rate of anal fin metamorphosis. Sp' Sr males passed from stage two to stage six in four weeks, whereas N'Sr males required 6.5 weeks for this process (Table 6). N' Sr males were from 30 to 60 percent larger than their Sp' Sr or Dr Dr siblings.

Crosses resulting in all-mule progeny consisting of four genoltypes: Four crosses were designed with X-Dr X-N' (ped. 3060,3065) or X-Sp' X-N' females (peds. 3209,321 1 ) and Y-Zr Y-Br males to yield all-male progenies that were comprised of four pigment classes, each having its own P genotype (Table 7). Two of these genotypes were particularly important. N' Br males carried two "late" P factors that were not necessarily identical. The age at which N' Zr males attained stage six had a bearing on the problem discussed above, namely whether the P factors on Y-Sr and Y-Zr are identical. It should be recalled that N' Sr males reached maturity sometime after 27 weeks of age (Table 5).

Sp' Zr and Dr Zr males became sexually mature earlier than males of any of the other three genotypes. N' Br males were the latest to reach stage six. Males


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TABLE 6

Rate of anal fin metamorphosis (stage 2 to stage 6) in male platyfish of different genotypes

91

Data from Pedigree table

3255 9 3255 9 321 1 7 3131 3 3537,-38 5 3537,-38 5 3073 5 BP 2 3131 3 321 1 7 321 1 7 BP 2 321 1 7 BP 2 3073 5 3537,-38 5 3537,-38 5

n

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Pattern

Dr Sp9 Spl Ir SpI Ir Spl Sr S p l Sr SpI Sr SpI Sr Ir Ir Sr Sr SpI Br NI Ir Ir Br NI Br Br Br NI Sr NI Sr NI Sr

P Genotype

PI P' P' P3 P' PJ PI P2 P' P2

P' P2

P' P2 P3 P3 P2 P2 P' P4 P5 P3

P3 P4 P5 P4 P4 P4 P5 P2 P5 P2

P5 P*

Age at stage 6

(weeks)

8 9

10.1 11.4 12.6 14.6 13.3 12.5 13.5 15.7 15.8 20.0 21.8 26.5 27.7 28.1 32.9

Stage 2 to stage 6 Treatment weeks s.e.

1 3.2 f 0.08 1 3.3 f 0.14 i 3.4 -C 0.13 i 4.0 f 0.10 i 4.0 f 0.30 m 4.3 f 0.14 m 4.2 f 0.21 m 3.8 -+: 0.14 i 4.2 -+ 0.10 1 4.8 f 0.11 1 4.9 f 0.14 m 5.0 f 0.19 i 6.5 f 0.46 m 6.5 f 0.20 m 6.9 f 0.30 i 7.7 -C 0.47 m 7.1 -C 0.32

TABLE 7

Age of sexual maturity and adult size of male platyfish of different genotype

Stage 2 Stage 6 Genotype n Age Size Age Sizeb

Dr Ir Dr Br NI Ir NI Br

Dr Br NI Ir

SpI Ir SpI Br NI Ir NI Br

SpI Ir SpI Br NI Ir NI Br

ped. 30600 8 5.4 f 0.3

10 9.7 k 0.4 7 12.4 f 0.8 7 12.6 f 1.0 ped. 3065c 2 10.5 f 0.4 2 11.5 f 1.1 ped. 331 IC

15 6.6 f 0.2 12 10.8 f 0.2 7 10.9 f 0.3 8 15.3 f 0.3 ped. 3209e

14 8.2 +. 0.2 5 14.0 & 0.6

10 14.0 f 0.5 7 18.4 k 1.5

17.6 & 0.6 24.6 f 0.5 27.9 f 0.8 30.9 f 1.1

24.0 f 0.5 24.5 f 0.5

d

d

9.1 -t- 0.2 14.9 f 0.6 17.6 k 0.8 22.9 rfr 1.7

15.0 0 17.5 3z 0.5

10.1 3z 0.2 15.7 5 0.3 15.8 f 0.3 21.8 3z 0.6

12.1 zk 0.3 19.4 f 0.6 19.3 zk 0.3 29.3 & 1.8

22.6 t 0.6 30.7 f 0.7 33.6 f 0.7 40.3 f 1.1

28.5 f 0.5 32.5 k 0.3

26.5 k 0.3 33.9 & 0.3 34.1 f 0.6 39.0 f 0.4

26.1 f 0.3 32.4 f 0.5 34.3 f 0.5 39.3 =k 0.7

~~ ~~ ~~

a Parental crosses are listed in Table 4. For pedigrees 3060 and 3065, this represents the size at which the males reached stage 2 and

stage 6; for pedigree 3211 this is adult size a t week 23, and for pedigree 3209, adult size at week 38. Each fish raised in isolation from day of birth.

d Not recorded. e Fish raised in two tanks: 1: 9 SpI Ir, 1 Spl Br, 5 NI Ir, and 3 NI Br;

2: 5 SpI Ir, 4 SpI Br, 5 NI Ir, and 4 NI Br.

92 K. D. K A L L M A N AND V. BORKOSKI

of genotypes Dr Br or Sp' Brand N 1 Zr were intermediate. Within each pedigree, the differences in age of maturity between Sp' Zr or Dr Zr males and the intermediate classes were highly significant ( p < O.Ol), as were the differences between the intermediate genotypes and N 1 Br males. It is important to note that the Sp' Zr and Dr Zr males attained stage six at a comparable age as males of the same genotype but belonging to different pedigrees (Table 3). There was no difference in age of maturity between N' Zr and Sp' Br males (ped. 3209, 321 1 ) , but N' Zr males of ped. 3060 matured somewhat later (1 7.6 weeks) than their Dr Br sibs (14.9 weeks). This difference is significant ( p = O.Ol), but it cannot be taken as evidence that the P factors linked to X-Sp' or X-Dr are different. Note that the N' Zr males of ped. 3060 reached stage two and stage six two weeks later than the N 1 Zr males of ped. 321 1. It is this two-week delay that accounted for the difference between Dr Br and N' Zr males in ped. 3060.

The well-marked differences between early, intermediate and late genotypes were present regardless of rearing conditions. In mass cultures, maturity (stage six) of the early genotypes was delayed by two weeks (20% longer than fish raised in isolation) and that of the late N' Br class by approximately seven weeks (34% 1 f

Within each pedigree, the initiation of stage two of the four genotypes was always in the same age sequence as the attainment of stage six. However, while N 1 Br males raised in isolation became sexually mature 11-12 weeks after the Sp' Zr or Dr Zr classes, they differed in the time of onset of stage two by only 7-9 weeks. This again signifies that the rate of anal fin metamorphosis was influenced by the P genotype and, furthermore, that the late classes required a longer period of time to pass from stage two to stage six than did early-maturing genotypes (Table 6). This relationship held true regardless of whether the fish were raised in isolation or in mass culture. The relatively slow rate of gonopodial development of N' Br males affected all stages, but appeared most marked in stage two. Of particular interest was the observation that in one pedigree, 3060, N' Zr and N 1 Br males entered stage two at approximately the same age, although the latter were more variable. The former reached stage six in 5.1 weeks, whereas the latter took twice as long. The transition from stage one to stage two was rapid and most pronounced in early-maturing males, while in late genotypes it was sometimes protracted and at times difficult to define over a period of one week.

The differences in age of sexual maturity were paralleled by differences in adult size. The early-maturing classes (Dr Zr or Spl Zr) of each pedigree were significantly smaller ( p < 0.01) than the two intermediate genotypes, which in turn were significantly smaller than the late-maturing males, N 1 Br ( p < 0.01). The data for pedigree 3060 also provided evidence that the early- and late- maturing genotypes entered stage two at different sizes (Table 7).

Contrary to many general statements in the literature concerning the cessation of growth of poeciliid fishes at maturity, which often have been poorly documented, males of X . maculatus continue to increase in size after reaching stage six. The growth increment, however, depended upon the P genotype (Table 8).

GENETIC CONTROL O F SEXUAL MATURITY

TABLE 8

Growth of males of X. maculatus, belonging to four genotypes, after attaining sexual maturity (ped. 3060, from Table 7)

93

Size (mm) at: Maturity Maturity + Increment since

Genotype n (from Table 7) 10 weeks 52 weeks 156 weeks. Maturity (Range)

Dr Ir 8 22.6 f 0.6 25.4 27.6 30.3 t 0.7 7.7 (5.5-10.5)

NI Ir 7 33.6 k 0.7 35.7 36.7 38.9 5 0.9 5.3 (3 .0~ 8.0) Dr Br 10 30.7 0.7 32.6 33.9 35.7 5 0.9 5.0 (4.5- 7.0)

NI Br 7 40.3 1.1 41.5 42.3 46.0 3- 0.9 4.7 (1.5- 5.5)

a By this time two Dr Ir males had died, as well as one fish each of the remaining three genotypes.

At three years of age, Dr Zr males had grown as much as 34 percent beyond their size at maturity. On the other hand, the corresponding increase for their late maturing N' Br sibs did not exceed 11.6 percent. Dr Br and N' Zr males were intermediate.

CoatzacoaZcos stock ( C p ) : Routine observations on the Cp stock had revealed that males with Y-Nz and Y-Spg matured under mass-culture conditions between eight and ten weeks and were of smaller adult size than males of any other platyfish stock. This suggested that the P factors on these two Y chromosomes might be earlier than those located on the Y chromosomes of the Jp and Bp stocks marked by Zr and Sr, respectively. Cp males with still a third Y chromosome marked by A r were consistently large than those without A r .

Comparison of Y-Spg of C p with Y-Ir of Bp: Two broods of X-Dr Y-Spg and X-Dr Y-Zr males (ped. 3255, Table 9) were raised in isolation. Sp9 males of both broods matured six to ten days earlier than their Zr sibs (first brood: SpS, 54.6 f 1.9 days; Zr, 60.9 -C 1.4 days; second brood: Sp9, 55.5 f 1.0 days; Zr, 66.2 f 3.2 days). The two classes of males also differed significantly in the onset of stage two, that is by five to ten days. Sp9 males reached maturity at a smaller size than Zr males ( p < 0.01). X-Dr Y-SpS males attained stage six at an earlier age and required less time for anal fin metamorphosis (3.2 weeks) than did males of any other genotype (Table 6).

Comparison of X-Spl of Jp with Y-N2 of Cp: Males of genotype X-Sp' Y-Zr and Y-N" Y-Zr differed neither in age of maturity nor size (ped. 3281 , Table 9 ) . Moreover, the values for these two genotypes were identical with those of the X-Dr Y-Zr class o f ped. 3255 (see above). The P factors on X-Dr, X-Sp', Y-N" and Y-Spg might be identical.

Comparison of X-Spl of Jp with Y-Ar of Cp: The results of ped. 3166 (Table 9) established that males with Y - A r matured significantly later than those with Sp9 (Sp' Sp9: 9.9 f 0.3 weeks; Sp9 A r : 21.0 2 1.5 weeks). Note that the Sp' Sp9 males had the same P genotype (the earliest P genotype known in males) as the Dr Spg platyfish of ped. 3255 (see above) and that the Sp' Sp9 males matured at a younger age than any other genotype of males raised under mass culture

94 K. D. KALLRIAN A N D V. BORKOSKI

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conditions. The P factor linked to Y-Ar of Cp resembled those linked to X-N' and Y-Br of Bp. This was also shown by the results of ped. 31 16.

Comparison of Y-Ir of B p with Y-Ar of C p : Sp9 Zr males of both broods cd ped. 3195 matured significantly earlier than their Sp9 Ar sibs (Table 9). This firmly established that the P factors linked to Y-Zr and Y-Ar were not identical. Further supportive facts were shown by ped. 3237 in which Zr Ar and Ar Ar males were compared with each other. Additional evidence that Y-Ar carries a P factor that is later in its expression than the one on Y-Zr of Bp was provided by the exami- nation of the females of the second brood of ped. 3195, sacrificed at ten weeks. Nine of 14 W-+ Y-Zr females contained yolky eggs whereas none of the ten W-+ Y-Ar females showed any signs of vitellogenesis. The two genotypes, however, did not differ in size (Zr: 20.0 f 0.2 mm; Ar: 19.4 * 0.1 mm; p = 0.1).

11. Females Females with and without N1: The onset of sexual maturity in females was

analyzed in great detail in one system in which females homozygous for an early P factor (X-Sp' X-Dr or X-Dr-X-Dr) were compared with homozygous late- maturing females (X-N' X-N') and heterozygoltes (X-N' X-Dr) . These females were obtained from three sets of crosses designed to yield experimental groups consisting of either heterozygous and homozygous early females (Table I O ) or heterozygous and homozygous late-maturing females (Tables 11, 12). For the

TABLE 11

Sexual maturity in females of pedigree 3153a

Month Age of

(weeks) birth

20 2 22 8 24 4 26 6 28 7 30 3

n - 10 6 9

11 14 10

Mature females size (mm) No. of yolky eggs, range

33.0 k 0.920 37.6 k 5.4 (5-61) 36.8 k 0.9@ G.0 t 4.5 (29-56) 29.3 f 0.57e 12.4 f 2.2 (7-23) 39.4 -I 0.67' 40.8 C 4.9 (12-60) 39.5 +- 0.5B 37.6 t 5.4 (9-82) 41.3 k 0.79h 55.5 f 5.9 (16-77)

Immature females n size (mm)

6 34.4 c a.& 7 34.9 t 0.89 12 30.3 C 0.69 10 39.7 t 0.52 5 39.7 f 0.20 3 42.2 k 0.73

Percent immature Pb

37.5 0.45 53.9 0.95 57.1 0.65 47.6 0.96 26.3 0.06 23.1 0.09

I* These females consisted of two genotypes, X-Dr Sd X-CPo NI and X-CPo N1 X-CPo NI, but since the Dr and CPo patterns interfered with each other and Sd showed less than 100% penetrance (see KALLMAN 1970b), the two genotypes could not be told apart. Only 11 out of the 103 females exhibited Sd and these were the only females known to be heterozygous for NI. Genetic ancestry of this pedigree is listed in Table 4. The females were raised in isolation from birth except for the experimental group which was sacrificed at 24 weeks.

Probabilities that ratio of mature to immature females deviates significantly from unity. 0 Four females with Sd: 27.0 mm 25 ova

34.5 52 35.0 35

d Two females with Sd:

e One female with Sd: f One female with Sd: g One female with Sd: h Two females with Sd:

35.5 45 35.0 36 38.0 55 29.0 18 36.5 44 37.0 42 38.0 57 43.0 77


initial attempt to determine the onset of sexual maturity of X-N' X-N' females, a cross was set up that resulted in X-Dr Sd X-CPo N' and X-CPo N' X-CPo N' fish (ped. 3153, Table 11). Note that the same sex chromosomes and P factors were involved as in the females listed in Table 10. As already discussed in MATERIALS AND METHODS, however, the expression of Dr was obscured by CPo, a factor closely linked to N', and Sd, a factor closely linked to Dr, was phenotypically expressed in only eleven females of ped. 3153. For this reason, homozygous and heterozygous females could not be told apart from each other except in the eleven Sd fish. Nevertheless, the results of ped. 31 53 have been included, because they provided evidence that homozygous N' N1 females matured much later and at a larger size than heterozygous N' females.

The results of ped. 3153, however, left unanswered the question as to the precise age at which N' N' females attained sexual maturity. Therefore, a third series of experiments (Table 12) was designed to obtain heterozygous X-Zy X-N' and homozygous X-N' X-N' females that could clearly be distinguished phenotypically (peds. 3436, 3444. 3445, 3446, 3447). By necessity, we had to intro- duce an X chromosome with an early P factor and a suitable marker gene, Zy, that had not yet been used in any of the experiments described above (admittedly a disadvantage), This X-Zy was derived from the Papaloapan stock and was the only such chromosome in our collection that would provide a suitable marker. The P factor linked to X-Zy is an early one, but whether it is identical with the one located on the X chromosomes of the Jamapa stocks is not known at this time.

Sp Dr, Dr Dr uersus Spl NI, Dr N' ? o : We have listed in Table 10, by age and genotype, the number and percentage of mature females, their standard length, and the number of yolky eggs per mature female. The fish of each experimental group (horizontal line) were litter mates that were raised under idmtical conditions. Differences between experimental groups reflect variations in rearing procedures, and these explain why in several instances younger fish may be of larger size and may contain a higher frequency of mature individuals than older age groups (e.g., Dr N' ? ? , born in January and May and sacrificed at eleven and 17 weeks, respectively).

Females without N' matured significantly earlier and at a smaller size than their N' sibs. All Sp Dr females ( n = 34) , belonging to six experimental groups, were mature, whereas all the N' sibs (n = 21) possessed undeveloped gonads (ped. a: age groups 9, 11, 12: 13, 15 weeks; ped. b: age group 14.5 Table 10). In five additional experimental groups, a significantly higher percentage of S p Dr females ( n =44, 73%) than Dr N' females ( n = 47, 9%) were mature (ped. a: 17 and 22 weeks; ped. b: 12, 14 and 18 weeks). In no experimental group did the frequency of mature N' females ever exceed that of the Sp' Dr class.

Dr Sp' and Dr N' females within the same experimental group did not differ significantly in size (one exception, age group 19, p = 0.04), but after 14 weeks of age, the mean length of the Dr N' class was usually somewhat larger than that of their Dr Sp' tank mates (seven experimental groups: b-7 week 14, a-7 week 15, a-5 week 17, b-4 weeks 18 and 19, a-6 week 20, a-2 week 22). The three pedi-

100 K. D. KALLMAN AND V. BORROSRI

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grees sacrificed during weeks 33 and 34 were originally raised in five aquaria and measured during weeks 22 and 23 (see footnote g, Table I O ) before being redistributed or combined. These data, too, showed that within experimental groups, Iv* females were usually somewhat larger than Dr Sp' sibs (significantly so for e. tank one).

The relationship between genotype, age and size at which the two classes of females developed ripe ova is illustrated in Figures 1 and 2. As early as eight

25

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IO

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Size, m m FIGURE 1.-The relationship between size, age, and sexual maturity of Sp' Dr and Dr Dr

females. The data have been taken from Table IO. Not listed were 19 mature females of age groups 33 and 34 that ranged in size from 31 to 39 mm. Most females became mature at 21 mm, irrespective of age. 0 = immature, = mature.


25

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weeks of age, yolky eggs were found in Dr Sp' females of 23.6 mm (range 23- 25). This appeared to be the youngest age for maturity in this genotype, since siblings sacrificed one week earlier at 22.7 mm (range 22-23) were all immature. From the eleventh week onward, females of this genotype below a minimum size (range 19-21 am) irrespective of age did not attain sexual maturity. The immature fish of each age group were usually distributed around the lower end of the size spectrum. Our results provided no evidence for a decrease of this minimum size for maturity with age.

104 K. D. K A L L M A N A N D V. BORKOSKI

Females of the genotype Dr N' reached sexual maturity as early as eleven weeks, provided that they had attained a size of at least 29 mm (Figure 2 ) . With increasing age, Dr N' females developed rip2 ova at progressively smaller sizes. At 15 weeks the minimum size was 28 mm, at 16 weeks 26 mm, at 18 weeks 25 mm, and at 25 weeks some females as small as 23 mm were sexually mature. As in the case with the Dr S@ sibs, the smaller fish of each age group lacked yolky eggs.

Within each experimental group up to 25 weeks of age and 30 mm standard length, Dr Sp' females possessed more ripe ova than their mature Dr-N' sibs, although the latter fish were somewhat larger. This difference was significant for a-6, week 20. Within both genotypes the number of ripe ova was more closely correlated with size rather than age (Table 10).

Dr N' and N* N1 ? ? : Although Dr N' and N I N 1 females could not be distinguished phenotypically, the results of pedigree 3153 (Table 11) pmvided strong evidence that NI NI females matured significantly later than N' Dr females and that some of the homozygous N 1 fish were still immature at 30 weeks. We assume that the immature fish represented the N 1 N' class, since the fish of this pedigree had surpassed by far the minimum critical size and age established for Dr N' females (Figure 2) . The fish of the experimental group (sacrificed at 24 weeks) that were raised under mass culture conditions, were significantly smaller and had fewer yolky eggs per ripe female than those raised ia isolation. Nevertheless, rearing conditions had no appreciable effect on the frequency of mature females.

If mature and immature females did indeed represent the Dr N' and N' N' genotypes. respectively, they should have occurred in equal frequencies. The deviation from unity was not significant for any experimental group, but there was a decline of immature fish in the two oldest age groups (Table 11 ) . More- over, the eleven females, in which the Sd pattern developed, were the only fish definitely known to be heterozygous and all eleven had yolky eggs. These observations, however, did not rule out the possibility that some of the mature fish could have been N 1 NI. Of all the immature fish, in one (28 weeks, 39.5 mm) vitellogenesis was just beginning as indicated by the presence of four greatly enlarged oocytes. Since this female had exceeded by more than 14 mm the minimum critical size for maturity of Dr N' fish, we presume that it was homozygous for NI. Since vitellogenesis may be completed within a period of only seven days (SCHREIBMAN and KALLMAN 1977), some N' homozygotes could have been included among the mature fish.

Iy N' uersus N' N1 0 0 : As in the previous experiments, all females belonging to the same experimental group comprised litter mates that had been treated in an identical manner. The two genotypes of a number of broods were sacrificed at different ages, because it became apparent during this investigation that by week 20, the Zy N' females had reached sexual maturity; there was little information to be gained by maintaining them much beyond that age. Their N' N' sibs, however, did not develop yolky eggs until some time after 34 weeks of age. Some of the females of both classes were raised in isolation to obtain


fast-growth rates in order to compare females of widely varying sizes in a number of age groups. According to Figure 2, a considerable size range would have been required to detect a possible relationship between size, age and sexual maturity within any particular genotype.

Females of genotype X-Zy X-N’ reached sexual maturity 20 weeks earlier and at a much smaller size than their X-N’ X-N’ sibs (Table 12). Beginning with week 13, most Zy N’ females possessed ripe ova provided that they had attained a size larger than 25 mm (with 4 exceptions). Regardless of age, only 59 percent of the fish of the 21 to 24 mm size classes ( n = 32) had attained sexual maturity (Figure 3). There was no indication of a decrease in this minimum size range for maturity with increasing age (compare age groups 14 and 20 weeks, Figure 3). As with the Dr-Sp’ and Dr N’ females, it was mostly the smallest fish of an age class that were immature. The number of ripe eggs per mature female was again more closely correlated with size rather than age.

The homozygous N’ N’ females did not develop yolky eggs until some time after 33 weeks of age. The only experimental series in which all of these females possessed ripe gonads was the one sacrificed at 104 weeks, but the sample size was too small to determine whether in fact all N’ N‘ females are mature at this age. There was no evidence for a minimum size for sexual maturity; in contrast to the three other genotypes of females examined, the immature N’ N’ fish of any age group were not concentrated near the lower end of the size spectrum, but instead were scattered throughout the range of sizes (Figure 4). This is well illustrated by the 42-, 44- and 55-week old age groups. There initially appeared to be a slight increase in the frequency of mature females with age; only three out of 18 fish had matured between 34 and 38 weeks of age as compared with ten out of 15 females 39 to 40 weeks old. Between 40 and 60 weeks, the percentage of females with ripe gonads did not increase with age, well documented by a comparison of the fish 42 and 55 weeks old. Beyond week 60, there was again an increase in the frequency of mature fish. Once again, as with the other genotypes of females, the number of eggs per ripe female was more closely correlated with size rather than age.

In discussing size of Dr Sp’ (early) and Dr N’ (late) females, we noted that Dr N’ females were somewhat larger than their Dr Sp’ tank mates after the latter had reached sexual maturity. A similar relationship existed between Zy N’ (early) and N’ N’ (late) females. Until week 19, Zy N1 and N‘ N‘ females of the same experimental groups did not differ in size in any consistent manner. Beginning with week 20, however, homozygous N’ females in each of the eleven relevant experimental groups were somewhat larger than their Zy N’ sibs (significantly so for age groups 25 e-12, 30 d-2, and 104 e-7; two-tailed T test). If the null hypothesis is changed to “NI N’ females are larger than their Zy N’ sibs after twenty weeks, because the Zy fish are committed to the use of part of their energies for yolk production rather than for body maintenance and growth,” a one-tailed T test can be used, and the difference in sizes also becomes significant for experimental groups b-12 (week 30), p = 0.04; a-5 (week 31), p = 0.03; c-12 (week 38) , p = 0.03; and b-5 (week 60), p = 0.04.


IO

.

. . 0: . .

.

20 30 40 Size , mm

FIGURE 3.-The relationship between size, age, and sexual maturity of Zy NI females. The data have been taken from Table 12. Most females became mature between 21 and 24 mm. 0 = immature, = mature.

100

70

60

m

f 50

e

s e

2

40

30

2c

IC


0 0 .

-mo . 0 . .

0 0 ) 0

w 000 0 0 . 0

0 . ) . 0 . ow ... 8 8 0

0 0 .

0 0 0

o g 0 0

g g o o o 0 8 0

0 0 8

.

IO 20 30 40 50 Size, mm

FIGURE 4.-The relationship between size, age, and sexual maturity of NI NI females. The data have been taken from Table 12. Some females became mature around 34 weeks of age. Size was of no crucial importance for the initiation of vitellogenesis. Immature fish of any age groups were not concentrated near the lower end of the size spectrum, as in the other genotypes. 0 = immature, = mature.

108 K. D. KALLMA1L- AND V. BORKOSKI


Correlation between size, age and number of ova within genotypes: For all genotypes there was a highly significant correlation between standard length and the number of ripe ova (Tables 13, 14). Age had no detectable effect on egg number in females homozygous for early ( D r Sp') maturation. Egg number, however, to a small degree was negatively correlated with age in the two genotypes heterozygous for NI, and strongly so in the N' N' homozygotes. The biological explanation of the negative correlation coefficient, ray.$ (Table 14), is that fish that attained a certain size at a relatively early age have more eggs than fish that reached the same size at an older age. From our data, we find that Dr N' females in the 28 to 30 mm range (TI = 8) had a mean number of 21.3 eggs at 13 to 15 weeks, but only 15 eggs at week 31 ( n = 8) . Comparable data for the 32 to 34 mm size class were 25 eggs ( n = 8) and 23.3 eggs ( n = 8) respectively. Zy N' females in this size range averaged 23 eggs when sacrificed between eleven and 15 weeks (n = 8) , but only nine eggs when examined at week 25 ( n = 10).

The regression coefficients of D r Sp', Zy N' and N 1 N' females did not differ significantly from one another. Since Dr Sp' females initiated vitellogenesis at a smaller size than did the other two classes, they always contained more ova at any given size than the Zy N' and N' N' females, in this order.

A different relationship existed between Dr Sp' and Dr N 1 females, because the N' class had a significantly higher regression coefficient. Dr Spl females commencing vitellogenesis at a smaller size and lower age than D r N' females had significantly more eggs up to 28 mm of standard length. Above that size (28-40 nun) the difference between the two classes disappeared. Females larger than 40 mm were not available for analysis.

DISCUSSION

Number of aZleZes: At least five alleles exist at the P locus that control the onset of sexual maturity by determining when the gonadotrops of the pituitary gland differentiate and become active (see Table 1). Among the P factors identified, those associated with the earliest maturation are located on the X chromosomes of the Jp stocks, Jp 163 A and B, X-Dr and X-Sp', and on the Y chromosomes of stock Cp 2840, Y-N2 and Y-Spg. The P factors on these four chromosomes appear to be identical and are denoted as PI. Under comparable conditions, males and females carrying any two of these chromosomes (X-Dr Y-Spg, ped. 3255, Table 9; X-Sp' X-Spg, ped. 3166, Table 9; X-Sp' X-Dr, ped. 3145, Table 10) matured earlier and at a smaller size than platyfish of any other genotypes. The Y chromosomes of the two Jp 163 stocks, Y-Sr, carry Pz, and a Y chromosome of the Bp stock, marked by Zr, carries Ps. Two different late P factors are located on an X and Y chromosome of the Belize stock. These are denoted as P4 (Y-Br) and P5 ( X - N 5 ) , respectively.

Critical crosses that showed P' and P$ to be different are listed in Table 3. They made it possible to compare X-Sp' Y-Sr males with Y-Sr Y-Sr fish (ped. 3131) and X-Sp' Y-Zr males with Y-Sr Y-Zr males (ped. 3134). The association of PI with the X chromosome and of P2 with the Y-Sr chromosome of the Jp 163


stocks must be long standing, presumably dating back to their introduction into the laboratory in 1939. In the early 1 9 5 0 ' ~ ~ MYRON GORDON brought some of these Jamapa fish to CURT KOSSWIG'S laboratory in Istanbul, from where they were disseminated further to other institutions in Germany. From one of these laboratories, ANDERS and ANDERS (1963) have reported that Jamapa males homozygous for Sr ( Y Y ) were larger and matured three to four weeks later than Sp' Sr or Sd Sr ( X Y ) males.

Differences between P' and Ps were documented in peds. 3229 and 3200 (Table 3), in which Sp' Zr and Zr Zr males were tested against each other, and in ped. 3255 (Table 9), which provided a comparison of X-Dr Y-SpS with X-Dr Y-Zr males. The data from the Belize stock (Table 2) demonstrated that P3 and P4 were distinct (Zr Zr tested with Zr Br and Zr Br with Br Br ) . Additional evidence was provided by comparing PIPs (X-Dr Y-Zr, or X-Sp' Y-Zr) with PIP4 (X-Dr Y-Br or X-Sp' Y-Br) males and P P 3 (X-N' Y-Zr) with P5P4 (X-N' Y-Br) males (Table 7). The crosses listed in Table 7 also showed convincingly that P' and P5 cannot be identical, and this was also demonstrated in Table 5, in which P'F and P5Pz males were compared with each other.

The action of Ps and PJ is similar in the sense that males homozygous or heterozygous for these two alleles matured at roughly the same age and size. The difference between the two alleles, however, was strikingly documented by their interaction with P5; males of genotype P5P* (X-N' Y-Sr) reached sexual maturity between 27 and 30 weeks at 38 mm (Table 5 ) , while P5PJ (X-N' Y-Zr) males reached stage six between 17 and 19 weeks at 34 mm (Table 7). Although comparisons of genotypes within experimental groups are more critical, we are convinced that these observations reflect genetic differences and are not due to variations in rearing conditions between broods, since both groups were reared in individual aquaria. We note that there were three pedigrees of N' Sr males (n = 54) and four pedigrees of N' Zr males (n = 26), and that comparisons within genotypes, but between pedigrees and treatments, showed no important het- erogeneity of results. Moreover, P'Pe males (Dr Sr, Sp' Sr) raised together with the N' Sr males, matured at similar ages as PIP2 fish of other pedigrees, thus serving as internal controls. The same held true for the PIP3 siblings (DrZr, Sp' Zr) of the P5P3 males.

No direct comparisons exist within pedigrees to determine whether P' is different from P4, P2 from P4 and P4 from P5. P3 has already been shown to be distinct from both P' and P4 (see above). Since PIPs males matured earlier and at a smaller size than P3P3 males (peds. 3229,3200, Table 3), which in turn reached stage six earlier than PsP4 males (Table 2), it follows that P' and P4 are distinct. We assume that Pe (Y-Sr) is different from P4 (Y-Br) , because homozygous Sr males became sexually mature ten and 15 weeks (Table 3) , but homozygous Br males not until 26.5 weeks.

The evidence that P4 and P5 are distinct is based not only upon comparisons between pedigrees, but also between males and females. Homozygous Br males (P4P4) became sexually mature around 26.5 weeks, while fifty percent of the

1 I 2 K. D. KALLMAN AND V. BORKOSKI

homozygous N 1 females (P5P5) still liad undeveloped ovaries at week 50 (Table 12, Figure 4). Rearing conditions could not be responsible for the delay of maturation of NI N’ fish, because many of them were raised under superior conditions resulting in a size that was far above that reported for females collected from natural populations ( GORDON and GORDON 1954). Differences between P4 and P5 were also indicated by a comparison o€ genotype P2P4 (Y-Sr Y-Br, ped. 3276, Table 3) , which reached stage six at 14 weeks and 30 mm, with PsP5 males (X-NI Y-Sr) (Table 5), which consistently matured between 27 and 33 weeks at 35 mm.

The P factors on the W chromosomes of the Cp and Bp stocks and on X-Zy of Pp are “early” factors relative to P4 and P5, but their precise identity is still unknown. W-+ Y-Zr and W-4- Y-Br females of the Bp stock became sexually mature at approximately the same age as Y-Zr Y-Zr and Y-Zr Y-Br males, respectively (SCHREIBMAN and KALLMAN 1977). Since the regression of egg number on standard length for Zy N’ and Dr N1 females was significantly different (Table 14) , the early P factors on X-Zy and X-Dr do not appear to be the same.

No effort has yet been made to assess the variability of natural populations with respect to P factors, and there is no reason to assume that we have identified all of them. The late factors, P4 and P5, were accidentally discovered during our studies on the distribution of the W and X chromosomes and certain pigment genes within natural populations. P* and P3 were thought to be identical until their different interactions with P5 became known. Theoretically, there could exist several dozen P factors, each one stipulating maturity at slightly different ages or sizes. Most likely, the P factor on Y-Ar of Cp (Table 9) represents a sixth allele at this locus. It is different from PI (Spl) and P3 (Zr) (Table 9). It also cannot be identical with P2 (Sr) , since PIP2 and PIPs males matured at approximately the same age (Table 3), while Sp9 Zr (P’P”) males reached stage two five weeks earlier than Sp9 Ar males (ped. 3195, Table 9). Males homozygous for Ar became mature at a younger age than Br Br fish, making it unlikely that the P factors linked to Ar and Br (P4) are identical.

Males with later genotypes should be easily recognizable because their large size sets them apart from other males. Since adult size is an indirect record of the age at which males have reached stage six, one can obtain an admittedly rather rough indication of the frequency of late genotypes in natural populations. Platyfish in the field, however, rarely attain the maximum sizes realized in our laboratory studies. The mean size of adult males of seven major river systems in Mexico, Guatemala, and Belize ranged from 20.0 0.03 (n = 1331) for the Rio Papaloapan to 25.3 2 0.1 (n = 346) for the Rio Jamapa (GORDON and GORDON 1954). The mean of the PzP5 (NI Sr) males and P4P5 (NI Br) males exceeded the maximum lengths of males in five and six of the seven populations, respectively. There seems little doubt that these or similar genotypes are quite rare under natural conditions. The mean size of the males from the Rio Pap- aloapan approached that of the PIP’ males; obviously this genotype or others with similar effects must be common in this population. Males from the Rio


Jamapa were somewhat larger, and their size corresponded to that of PIP2, PsP8, P2P3, and P3P3 males. Our experimental data also indicate that most P genotypes of platyfish stipulate maturation at a relatively early age. Nine of the 15 genotypes analyzed brought about sexual maturation between eight and 15 weeks (Tables 15 and 16), two between 16 and 20, three between 21 and 30 weeks, and one at irregular intervals between 34 and 104 weeks. GORDON and GORDON (1954) found that in the Jamapa population males with sex-linked macro- melanophore patterns were significantly larger than those homozygous for the recessive (+) allele. Does this mean that P factors stipulating maturity at a somewhat larger size or older age are more frequently found linked to macro- melanophore genes than to the + alleles?

P4 linked to Br was discovered among a sample of 38 fish collected in Belize in 1966 and brought to the laboratory for breeding purposes. Two of the 76 chromosomes were marked by Br, and if both of them also carried P4 (one chromosome was not tested), its frequency would be 0.0263. P5 was found in a single fish out of a sample of 85 collected in Belize in 1969, giving it a frequency of 0.0059. Thus, at least two late factors are present in this population.

P genotypes and egg production: The P locus occupies a key position in the life history of platyfish. An interplay of a large number of factors must account for the maintenance of this polymorphism in natural populations. Females of genotype PIP' were mature at 21 mm, a size they can attain within 8 to 9 weeks under optimum conditions. Since they develop ripe eggs earlier than any other known genotype, they may be at a selective advantage under such conditions. As suggested by our data, these females can control the onset of sexual maturity to a limited degree by adjusting their growth rate in response to environmental factors, but once they have reached this critical size (21 mm), they initiate yolk production (Figure 1). Should trophic conditions deteriorate, mature PIP' females will be under stress and will have available less energy for body maintenance, unless gonadal regression is induced, than do immature females of equal size but different genotypes that determine a larger critical size for vitellogenesis (e.g., Sp' N' 0 0 ) . Such stressed females might be eliminated through disease or predation either before or after their first brood. Differences in energy utilization were probably responsible fm the larger size of the late maturing class of females (Tables 10 and 12). This effect was not noted when the two classes were raised individually in isolation (Table 11 ) .

Calories stored in the mature germ cells and those required for their production represent consumed food energy that is lost to the body for other purposes (PHILLIPS 1969). Females of Gasterosteus aculeatus, once committed to vitellogenesis, lost weight unless the cost of egg production was met by the amount of food consumed ( WOOTTON and EVANS 1976).

Heterozygous females, PIP5, may possess a genotype that affords them maximum flexibility to respond to environmental conditions. The minimum size at which these females were found to possess yolky eggs decreased with increasing age. Under optimum conditions those fish exhibit a fast growth rate and may

114 IC. D. KALLMAN AND V. BORKOSKI

TABLE 15

Age at sexual maturity of five homozygous genotypes of platyfish

P genotype

P' P' P' P' PI PZ PZ P2 PZ PZ Pf PS P4 P4 P4 P4 P5 P5

-___ Pigment pattern

Dr Sp9 Dr Spl Sr Sr Sr Sr Sr Sr Ir Ir Br Br Br Br NI NI

Sex Maturity

n weeks s.e Treatments

23 8b 8 4

12 17 46 3

16+

7.9 t 0.2 8.0

13.5 f 0.2 10.8 c 0.8 14.6 k 0.3 12.6 t 0.3 26.5 f 0.6 24.7 t 2.67

3 4 1 04

i m i

m m m

i, m

1

1

Pedigree from table

3255 9 3145 10 3131 3 3204 3 3131 3 Bp stock 2 Bp stock 2 Bpstock 2 many 12

i: fish raised in isolation; m: mass culture.

Some females still with undeveloped gonads at 73 weeks. b Fish are 23 mm in length.

TABLE 16

Age at sexual maturity of ten heterozygous P genotypes of platyfish

P Pigment genotype pattem Sex n

Maturity weeks s.e. Treatment" Pedigree from table

P' PI P' PS P' PS P' Pf P' P2 P' P4 P' P4 P' P4 P' P5 P2 P3 PZ PS PZ P4 Pa P5 P2 P5 PS P4 P3 P4 PS P4 PS P5 PS P5 PJ P5 P4 P5 P4 P5 P4 P5

Spl Sr 8 8 Dr Ir $8 SpI Ir $ 8 Spl Ir $ 8 Sp' Sr 8 8 Dr Br $ 8 Sp' Br $8 Sp' Br $ 8 Dr NI 0 O b Sr Ir $8 Sr Ir $8 Sr Br 8 8 Sr N1 $ 8 Sr NI 88 Ir Br $8 Ir Br $ 8 Ir Br 8 8 Ir NI 8 8 Ir NI 88 Ir NI 8 8 Br NI 88 Br NI 88 Br NI 88

15 8

14 12 20 10 12 5 5 3 9

16 7

14 19 40 11 7 7

10 7 8 7

10.1 c 0.2 9.1 t 0.2

12.1 t 0.3 10.8 +. 0.2 11.4 f 0.1 14.9 rfr. 0.6 15.7 f 0.3 19.4 rfr. 0.6 11.0 12.5 f 1.0 12.3 2 0.1 14.1 t 0.4 28.1 f 1.0 32.9 f 0.7 19.7 f 0.5 20.2 t 0.5 16.1 f 0.6 17.6 f 0.8 15.8 f 0.3 19.3 f 0.3 22.9 f 1.7 21.8 f 0.6 29.3 f 1.8

1

i m m i i 1

m m i m m i m m m m i i m i 1

m

321 1 3060 3209 3229 3131 3060 321 1 3209 3145 3204 3134 3276 3537,-38 3537,-38 Bp stock Bp stock 3276 3060 321 1 3209 3060 321 1 3209

7 7 7 3 3 7 7 7

10 3 3 3 5 5 2 2 3 7 7 7 7 7 7

i: fish raised in isolation; m: mass cultures. b Females must be 29 mm in length.


surpass their critical size for yolk production at eleven weeks, or three weeks after P’P’ females do. Under poor conditions, P1P5 females have a slower growth rate and may remain for 25 weeks below the minimum size for vitellogenesis (23 mm) (Figure 2). Up to this age and size, they can utilize all available energy for body maintenance and growth. Should conditions improve during this period, they can increase their growth rate and surpass the minimum critical size for vitellogenesis within two or three weeks. There are some indications that the minimum site for yolk production of W - f Y - Z r females may also decrease with increasing age (SCHREIBMAN and KALLMAN 1977). P5P5 females, which do not commence yolk production until some time between 33 and 104 weeks of age, are clearly at a disadvantage.

Late maturation was not offset by an increased number of eggs, in spite of the larger size of P5P5 females (Tables 13,14). For any given size, P5P5 females consistently had fewer eggs than females of any other genotype. PIP’ females enjoyed a double advantage over heterozygotes, PIP5, for they matured not only earlier and at a smaller size, but they also possessed more eggs than P1P5 females at sizes up to 28 mm. A possible heterozygous advantage of PIP5 females, however, was indicated by a projection of the regression curves into the 40 to 50 mm range. This predicted that above 40 mm, P1P5 females will contain significantly more eggs than females of any other genotype. Whether this has any real biological meaning, if indeed this could be verified experimentally, is doubtful in view of the fact that females larger than 40 mm are exceedingly uncommon in natural populations (GORDON and GORDON 1954). The mean size of females (n = 476) of the Rio Jamapa population was 25 mm, and none exceeded 40 mm.

Size is positively correlated with dominance in males of X . maculatus (BRAD- DOCK 1945; SOHN 1977), and in several other species of poeciliid fishes (BAIRD 1968; BOROWSKY, unpublished thesis; CONSTANTZ 1975; MCKAY 1971). This may have survival value in securing preferred access to a number of limited resources, thus offsetting the “cost” of a delay in sexual maturity. Larger fish are usually thought to be less preyed upon than smaller ones and can undoubtedly feed upon a more varied number of food items. Large dominant males of Poecilia lucida, which occur together with the all-female P. monacha-lucida complex, have preferred access to conspecific females (MCKAY 1971) , and dominant males of Poecilia reticulata inseminate more females and sire more progeny ( GANDOLFI 1971). Whether dominance can also be translated into greater reproductive suc- cess in X . maculatus is not known.

EfJects of size and age on maturity: The effects of size and age on maturity are difficult to separate, since as the fish grow older, they also increase in size. The question exists whether it is size, or weight, or a cue closely associated with either of these factors, rather than age, that controls the onset of gonadotrop activity. The evidence provided by fisheries’ statistics has shown that in most species size is more closely correlated with maturity than age (ALM 1959; BOWERING 1976; PINHORN 1966). ALM also found that for any given age of an experimental group or population, it was usually the smallest fish that had


undeveloped gonads, and this suggests that a certain size or weight was crucial for the initiation of maturity. A genetic effect was indicated since the crucial size was not the same for different stocks of the same species (ALM 1959).

The data from two of the four genotypes of female X . maculatus examined in detail conformed to this scheme rather well (Figures 1 , 3 ) . For PIP' females, the critical size was approximately 21 mm. Depending upon the growth rate, which was manipulated environmentally, these females became mature between eight and 25 weeks whenever they had reached 21 mm. For Zy NI females, the critical size fell at some point between 21 and 24 mm (Figure 3). But age was also sometimes a significant factor, for the critical size of P1P5 females decreased with increasing age (Figure 2). To describe the conditions under which these females initiated vitellogenesis required a statement both as to their size and their age. They were mature as early as eleven weeks provided they had reached at least 29 mm, but at 25 weeks even fish as small as 23 mm had yolky eggs. For P5P5 (N' N' ) females, there was no relationship between the presence of ripe ova and size. For most of their age groups, immature fish were scattered throughout the size range and not-as in the other three genotypes-concentrated at the lower end of the size spectrum (Figure 4) . Because P5P5 females were at least eight months old when they reached maturity, and grew at a faster rate than mature females, they far exceeded the mean size of platyfish females from natural populations.

Under controlled pond conditions, the Chinese race of the common carp Cyprinus carpi0 matured significantly earlier than the European race. At maturity the European race had also a proportionally smaller gonad size. The simi- larity of this situation to PIP' platyfish that mature at 21 mm and at eight weeks and have a high fecundity, and to P5P5 fish that mature sometime after 34 weeks of age at a much larger size and that have a low fecundity, is striking. HULATA, MOAV and WOHLFARTH (1974) suggested that differences between the two races had evolved in response to different culture conditions. In Europe, selection was for the biggest fish raised in monoculture at low population density with special rearing ponds for fry. In China, polyculture (many species) was practiced, often resulting in poor pond conditions. Under such a regimen, early reproduction and a high reproductive rate may have had a selective advantage.

Environmental conditions strongly influenced the age at which males matured, since a given genotype often reached stage six at a younger age when the individual was raised in isolation. In order to ascertain the relative importance of size (or weight) and age in determining the onset of gonadotrop function in males, a given genotype must be raised under at least two environmental conditions that result in different growth rates. The size (or weight) and age at which the males entered stage two should be recorded. This could be done for PIP" and P'P4 males (Table 17).

Considering the figures for fish that were reared in isolation as a base line, PIPs females raised under mass-culture conditions required 80 percent longer (4.5 weeks) to reach stage two, but were only 2.4 mm (18%) larger. For P'P4


TABLE 17

The effects of rearing conditions on size and age at stage two and stage six in two genotypes of platyfish

117

Pedigree Stage 2 Stage 6 and treatment Genotype Size, mm Age, weeks Size, mm Age, weeks T I

2974m PIP3 Sp1 Ir 20.3 t 0.3 10.0 t 0.5 22.3 C 0.3 14.2 t 0.3 6 2974 m PIPS Dr Ir 19.4 t 0.4 9.7 t 0.5 22.3 * 0.5 14.1 t 0.5 7 3060i PIPS Dr Ir 17.6 C 0.6 5.4 k 0.3 22.6 C 0.6 9.1 t 0.2 8

2974m PIP4 Spl Br 22.1 +. 0.3 13.2 C 0.5 27.0 -t 0.7 20.5 C 0.8 6 2974i PIP4 Dr Br 23.9 t 0.4 16.0 t 0.7 27.5 C 0.5 21.4 t 1.0 9 3060i PIP4 Dr Br 24.6 t 0.5 9.7 t 0.4 30.7 2 0.7 14.9 2 0.6 10

a Data for ped. 3060 from Table 7 and for ped. 2974 from KALLMAN and SCHREIBMAN (1973), slightly modified. Each male of ped. 3060 raised in isolaticm from birth in 8-liter tank; all 28 males of ped. 2974 raised in 33-liter tank.

males, mass cultures delayed the onset of stage two by 36 (Spl Br, 3.5 weeks) to 65 (Dr Br, 6.3 weeks) percent, but the size of PIP4 males at stage two, regardless of rearing conditions, did not vary appreciably. Since size was so much less variable than age, the attainment of a minimum size may be a significant factor fo r the onset of gonadotrop function in males, at least for the two genotypes examined (Table 17). We are aware that the comparison of ped. 3060 with 2974 is far from ideal, because we are dealing not only with two experimental groups exposed to different rations and tank conditions, but also because one group (2974) was exposed to intense social interactions, while the other was not. Social interactions are known to influence both age and size at sexual maturity in X . maculatus (SOHN 1977) and X . uariatus (BOROWSKY 1973), with sub- ordinate fish maturing later and at a larger size.

Possible sites of gene action: The genetic polymorphism at the P locus has a bearing on the problem of gene activation in higher vertebrates. Although the first effects of the P factors are observed in the pituitary gland, there is no com- pelling reason to assume that this represents the primary site of gene action. The gonadoltrops, in turn, are controlled by the hypothalamus and higher centers of the brain (PETER 1973) which may integrate and transduce peripheral information (weight, age or some factor associated with it) into an appropriate signal. Lesions in certain parts of the nucleus lateralis tuberis, but not in other areas of the hypothalamus, caused a significant decrease in gonadal size in Cmassius auratus (PETER 1970). This suggests that a quantitative relationship exists between gonadal development and the strength of a signal from the hypothalamus to the gonadotrops. The P gene may control the strength of such a signal, thereby accounting for the protracted sexual development of late-maturing males and the small gonadal size of later-maturing, P P 5 , females. The P factors could also control a metabolic product that is crucial for the differentiation of the gonadotrops, or they could control “sensors” either within the undifferentiated gonadotrops or elsewhere. The gonadotrops of late-maturing genotypes appear


to differentiate more slowly, once the signal for the initiation of sexual maturity has been received. The rate of anal fin metamorphosis is directly correlated with the age at which male platyfish entered stage two (Table 6) suggesting that adequate hormone titers for successive stages of gonopodial development are attained more slowly in the later genotypes (SCHREIBMAN and KALLMAN

1977). In typical Mendelian inheritance, a heterozygote with one “normal” and one

“defective” allele exhibits a more or less normal phenotype. If the P factors belong to a structural gene that specifies a metabolic product required for the onset of gonadotrop differentiation, one could expect heterozygotes to initiate activity at the same age as the genotype homozygous for the earlier allele. This was not the case, but some allelic interaction was indicated. The phenotypes of P2Pz, PzPs and PsPs males were virtually identical (stage six around twelve weeks, Tables 15,16), but PsP5 males attained stage six between 15 and 19 weeks and, by contrast, P2P5 fish did not become sexually mature until 27 to 30 weeks. Compared with P4 and P5, Pa must be considered an early allele (Table 15), but PzP5 males matured at the same time as those with the P4P5 and P4P4 genotypes. PzP4 males were similar to the Pz homozygotes. Thus, whether a gene is considered “early” or “late” depends in part upon the other P factor with which it is combined.

This work was supported in part by Public Health Service Grant GM-19934. We thank DR. JAMES W. ATZ, American Museum of Natural History, for reading the manuscript critically.

LITERATURE CITED

ALM, G., 1959 Connections between maturity, size and age in fishes. Rep. Inst. Freshwater

ANDERS, A. and F. ANDERS, 1963 Genetisch bedingte XX- and X Y - 0 9 und Y Y - 8 8 beim

BAIRD, R. C., 1968 Aggressive behavior and social organization in Mollienesia latipinlm Le

BOROWSKY, R. L., 1973 Social control of adult size in males of Xiphophorus variatus. Nature

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a sex-linked gene controlling the onset of … · a sex-linked gene controlling the onset of sexual...

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